Type I diabetes is an autoimmune disease of humans caused by destruction of pancreatic islet beta cells. At present the disease is irreversible, although its symptoms are controlled by the administration of exogenous insulin. Type I diabetes is one of the most common autoimmune diseases in human populations and is a major public health concern.
It has previously been found that transplantation of a whole pancreas or of isolated islet cells is an effective treatment for Type I diabetes to restore insulin independence, when combined with immunosuppressive therapy. The success of existing therapies with isolated islets from human cadaver donors is a proof in principle that a cell-based therapy for human diabetes can be successful. However, the lack of available organs or islet cells has restricted this therapy only to very selected patients. The amount of islet cells which can be harvested from human cadavers is extremely limited. Therefore, a technology that is capable of producing significant quantities of islet cells would be highly desirable with regard to potential therapies for this disease.
Primate and human embryonic stem cells (ESCs) have been isolated and proliferated in culture. Embryonic stem cells are stem cells that can be maintained indefinitely through self-renewal and proliferation in culture, but which also retain the ability to differentiate spontaneously into cells of many different lineages. Under nonselective conditions, it has been previously demonstrated that a wide variety of stem cells, including mouse and human ESCs, will differentiate spontaneously into cells of many lineages including the pancreatic lineage. It has been previously shown that such differentiated cells can express the pancreatic duodenal homeobox 1 (PDX 1) gene, a transcription factor specifying the pancreatic lineage and can also express the insulin hormone. However, without selective conditions, stem cells will spontaneously differentiate into a wide variety of different lineages and only a small proportion of the cells will be differentiated towards any particular lineage.
Culture systems that allow the spontaneous differentiation of hESCs into insulin-staining cells have been reported (Assady, S. et al., Insulin production by human embryonic stem cells. Diabetes 50, 1691-1697 (2001); and Segev, H. et al., Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22, 265-274 (2004)). However, these studies neither investigated endoderm marker expression nor demonstrated development of cells possessing stereotypical characteristics of β cells: simultaneous expression of C-peptide and pancreatic duodenal homeobox 1 (PDX1), which is required for pancreas formation and co-activates the insulin promoter (Jonsson, J. et al., Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371, 606-609 (1994)). Because non-β cells such as neuronal cells, may express insulin (Sipione, S. et al., Insulin expressing cells from differentiated embryonic stem cells are not beta cells. Diabetologia 47, 499-508 (2004)), and insulin present in the culture media may be taken up into other cell types under certain conditions in vitro (Rajagopal, J. et al., Insulin staining of ES cell progeny from insulin uptake. Science 299, 363 (2003)), it is important that the endoderm and pancreatic origin of insulin-staining cells derived from hESCs be ascertained.
It was recently reported that spontaneous differentiation of human ESCs produced PDX1+/FOXA2+ cells and co-transplantation of these differentiated cells with mouse dorsal pancreas (E13.5) resulted in PDX1+/insulin+ cells, and co-staining of insulin and C-peptide was observed (Brolen, G. K. et al., Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells. Diabetes 54, 2867-2874 (2005)). This report demonstrated that pancreatic lineage cells can be induced from spontaneously-differentiating human ESCs by signals emanating from the embryonic pancreas. However, the experimental methods would be impractical to adopt into a high-throughput culture protocol and the nature of the molecular signals was not revealed by this study. In addition, unselected stem cell populations are tumorigenic, meaning that they will generate non-malignant tumors, known as teratomas, in immunodeficient animals in the same way that undifferentiated ES cells will.
Several studies have evaluated the effects of growth factors on human ESC differentiation to endoderm (Schuldiner, M. et al., Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97, 11307-11312 (2000) and D'Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534-1541 (2005). Yet, the state of the art is that reproducible, highly efficient differentiation to pancreatic precursors and islet cells is not routinely achievable. Furthermore, insulin producing cells generated using previously reported methods are less responsive to glucose (i.e. less functionally mature) than adult human beta cells and are believed to possess a phenotype more like immature beta cells. Taken together, these studies indicate that additional signals may be necessary to convert endoderm into pancreatic progenitors and insulin expressing cells into maturely functional beta cells. Studies of growth factor regulation of pancreas development in embryo models may provide important insights for directing hESC differentiation towards the pancreatic lineage (Wells, J. M. & Melton, D. A. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development 127, 1563-1572 (2000)). For example, in a chick-quail chimera system, it was demonstrated that BMP4 induces pdx1 expression in uncommitted endoderm and noggin blocks pdx1 expression in committed endoderm (Kumar, M. et al., Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev. Biol. 259, 109-122 (2003)).
Methods have been discussed in the patent literature for the differentiation of human ESCs, or other human pluripotent cell types, into pancreatic or pancreatic islet cells. However, as the technology of stem cell culture moves ahead, improvements in the techniques to culture various differentiated cell types are critical to the ultimate commercial use of these differentiated cells. The previous techniques reported for culturing human ESCs into cells of the pancreatic lineage, while suitable for laboratory scale investigations, can not be readily scaled up to reliably and consistently produce large numbers of pancreatic cell types for research or for therapeutic uses. Thus, a simple, reproducible culture method utilizing defined components that promotes islet differentiation from human pluripotent stem cells is a desirable addition to the field.